Name: in vitro assessment of cardiac reprogramming by measuring cardiac specific calcium flux with a gcamp3 reporter
Uploaded: 2022-02-22T13:00:00
Description: Watch this Scientific Journal Video about In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter at JoVE.com

我们感谢Leo Gnatovskiy在编辑本手稿英文文本方面所做的努力。图 1 是使用 BioRender.com 创建的。这项研究得到了美国国立卫生研究院（NIH）（1R01HL109054）对王博士的资助。

所有涉及动物的实验程序和实践都得到了密歇根大学动物护理和使用委员会的批准。所有涉及细胞培养的实验程序和实践必须在无菌条件下进行BSL2生物安全柜。对于涉及病毒的程序和做法，遵循了正确处理转染细胞，移液器吸头和管子以避免环境和健康危害风险的指南。
1. 建立Tg（Myh6-cre）1Jmk/J /Gt（ROSA）26Sortm38（CAG-GCaMP3）Hze /J（称为Myh6-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3）小?...

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E., Kim, M. S., Bassel-Duby, R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/protein+kinase+B+enhances+transcriptional+reprogramming+of+fibroblasts+to+functional+cardiomyocytes.">Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (38), 11864-11869 (2015).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Mll1+H3K4+methyltransferase+activity+to+guide+cardiac+lineage+specific+reprogramming+of+fibroblasts.">Targeting Mll1 H3K4 methyltransferase activity to guide cardiac lineage specific reprogramming of fibroblasts.</a> Cell Discovery. 2, 16036 (2016).</li><li>Grant, A. 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评估iCM功能对于心脏重编程领域是必要的。在这份手稿中，该协议描述了已经建立的Tg（Myh6-cre）1Jmk / J / Gt（ROSA）26Sortm38（CAG-GCaMP3）Hze / J小鼠品系，如何使用从该菌株中新生小鼠分离的NCF进行重编程为iCM，以及通过Ca2 + 通量评估iCM功能。这是一种评估iCM功能成熟的 从头开始 的方法。
几个关键步骤对于使用此方法成功重新编程和评估非常重要。首先，...

心肌梗死 （MI） 是世界范围内的一种严重疾病。心血管疾病 （CVD） 是全球主要死因，2019 年造成约 1860 万人死亡1，2。在过去半个世纪中，心血管疾病的总死亡率有所下降。然而，在一些不发达国家，这一趋势已经放缓甚至逆转1，这需要对心血管疾病进行更有效的治疗。作为心血管疾病的致命表现之一，在美国，心肌梗死约占 CVD 死亡总数的一半2。在缺血期间，随着冠状动脉阻塞以及营养和氧气供应有限，心肌发生严重的代谢变化，损害心肌细胞（CM）的收缩功能，并导致CM死亡3。心血管研究中已经探索了许多方法来修复心脏损伤并恢复受伤心脏的功能4。直接心脏重编程已成为修复受损心脏并恢复其功能的一种有希望的策略5，6。通过引入Mef2c，Gata4，Tbx5（MGT），成纤维细胞可以在 体外 和 体内重新编程为iCM，这些iCM可以减少疤痕面积并改善心脏功能7，8。
虽然心脏重编程是心肌梗死治疗的一种有前途的策略，但仍存在许多挑战。首先，重编程效率、纯度和质量并不总是像预期的那样高。MGT诱导只能达到总CF的8.4%（cTnT+）或24.7%（αMHC-GFP +），以在体外7重新编程为iCM7，或在体内高达35%8，这限制了其应用。即使系统中诱导了更多的因素，例如Hand29或Akt1 / PKB10，重编程效率仍然勉强令人满意，无法在临床环境中使用。因此，该领域需要更多的研究来提高重编程效率。其次，iCM的电完整性和收缩特性对于有效改善心脏功能非常重要，但评估这些特性具有挑战性。目前，该领域广泛使用的评价方法，包括流式细胞术、免疫细胞化学、qPCR等一些关键CM基因表达，都集中在iCM和CM的相似性上，但与iCM的功能特征没有直接关系。此外，这些方法具有相对复杂的程序，并且非常耗时。虽然重编程研究通常涉及筛选促进iCM成熟的潜在重编程因子11，但心脏重编程需要一种基于iCM功能的快速便捷的方法。
在每个收缩周期中，CM打开细胞膜上的电压门控钙离子通道，这导致钙离子（Ca2 +）从细胞间液短暂流入细胞质以参与肌原收缩。这样的Ca2+ 流入和流出循环是心肌收缩的基本特征，构成了CMs12的正常功能。因此，检测Ca2 + 流入的方法可能是测量CM和CM样细胞（包括iCM）功能的潜在方法。此外，对于iCM，这种方法提供了另一种评估重编程效率的方法。
遗传编码钙指示剂（GECI）已被开发并广泛用于指示细胞活性，特别是动作电位。通常，GECI由Ca2 + 结合结构域（例如钙调蛋白）和荧光结构域（例如GFP）组成，而GCaMP3是具有高亲和力和荧光强度的结合结构域。当局部钙浓度改变时，GCaMP3的荧光结构域将被激活13。在本文中，描述了一种在Myh6 +细胞中特异性表达GCaMP3报告基因的小鼠品系。通过将MGT引入该菌株新生儿的分离NCF中，可以通过荧光监测重编程，成功重编程的iCM将表现出来。这种小鼠菌株和方法将为研究心脏重编程提供有价值的平台。

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			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plates</td>
			<td>Alkali Scientific</td>
			<td>TP9006</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Stemgent</td>
			<td>04&#8211;0014</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X800E</td>
			<td>Inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin S HCl (10 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113903</td>
			<td></td>
		</tr>
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			<td>Bovine Serum Albumin (BSA) DNase- and Protease-free Powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP9706100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.2 MicroBeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-101</td>
			<td>Thy1.2 microbeads</td>
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			<td>Collagenase, Type 2</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9693955</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-671-51B</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>11960069</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-040-133</td>
			<td></td>
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		<tr>
			<td>Ethanol, 200 proof (100%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
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		<tr>
			<td>Ethylenediamine Tetraacetic Acid (Certified ACS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>E478-500</td>
			<td></td>
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		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
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		<tr>
			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td></td>
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			<td>IMDM media</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td></td>
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		<tr>
			<td>IX73 Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX73P2F</td>
			<td>Inverted fluorescence microscope</td>
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		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-668-019</td>
			<td></td>
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		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199, Earle&#39;s Salts</td>
			<td>Thermo Fisher Scientific</td>
			<td>11150059</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS Separator and Starting Kits</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
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			<td>Millex-HV Syringe Filter Unit, 0.45&#160;&#181;m, PVDF, 33&#160;mm, gamma sterilized</td>
			<td>Millipore Sigma</td>
			<td>SLHV033RB</td>
			<td></td>
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		<tr>
			<td>MM589</td>
			<td></td>
			<td></td>
			<td>Obtained from Dr. Shaomeng Wang&#8217;s lab in University of Michigan</td>
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		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31-985-070</td>
			<td></td>
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		<tr>
			<td>PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-010-049</td>
			<td></td>
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		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
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		<tr>
			<td>Platinum-E (Plat-E) Retroviral Packaging Cell Line</td>
			<td>Cell&#160;Biolabs</td>
			<td>RV-101</td>
			<td></td>
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		<tr>
			<td>pMx-puro-MGT</td>
			<td>Addgene</td>
			<td>111809</td>
			<td></td>
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		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Millipore Sigma</td>
			<td>P5413-1KG</td>
			<td>PEG8000</td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Millipore Sigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>PTC-209</td>
			<td>Sigma</td>
			<td>SML1143&#8211;5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IGF-I</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>ST 16 Centrifuge Series</td>
			<td>Thermo Fisher Scientific</td>
			<td>75-004-381</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-363-547</td>
			<td>40 &#181;m strainer</td>
		</tr>
		<tr>
			<td>Surface Treated Tissue Culture Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090015</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Millipore Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>02215365</td>
			<td></td>
		</tr>
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</table>

in vitro assessment of cardiac reprogramming by measuring cardiac specific calcium flux with a gcamp3 reporter

心脏重编程已成为修复受损心脏的潜在有希望的疗法。通过引入多种转录因子，包括Mef2c，Gata4，Tbx5（MGT），成纤维细胞可以重新编程为诱导心肌细胞（iCM）。这些iCM在梗死的心脏 中原位 产生时，与周围的心肌在电和机械上集成，导致疤痕大小的减小和心脏功能的改善。由于iCM的重编程效率、纯度和质量相对较低，iCM的表征仍然是一个挑战。该领域目前使用的方法包括流式细胞术、免疫细胞化学和qPCR，主要集中在心脏特异性基因和蛋白表达上，而不是iCM的功能成熟。由动作电位触发，心肌细胞中电压门控钙通道的打开导致钙快速流入细胞。因此，量化钙流入速率是评估心肌细胞功能的有希望的方法。在这里，该协议介绍了一种通过钙（Ca2 +）通量评估iCM功能的方法。通过将Tg（Myh6-cre）1Jmk/J（下面称为Myh6-Cre）与Gt（ROSA）26Sortm38（CAG-GCaMP3）Hze/J（下面称为Rosa26A-Flox-Stop-Flox-GCaMP3）小鼠杂交，建立了αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3小鼠品系。分离来自P0-P2新生小鼠的新生儿心脏成纤维细胞（NCF） 并在体外培养，并将MGT的多顺式构建引入NCF，这导致它们重新编程为iCM。由于只有成功重编程的iCM才能表达GCaMP3报告基因，因此可以通过Ca2 + 通量和荧光显微镜直观地评估iCM的功能成熟。与未重编程的NCF相比，NCF-iCM显示出显着的钙瞬时通量和自发收缩，类似于CM。该协议详细描述了小鼠品系的建立，新生小鼠心脏的分离和选择，NCF分离，用于心脏重编程的逆转录病毒的产生，iCM诱导，使用我们的报告线评估iCM Ca2 + 通量，以及相关的统计分析和数据呈现。预计这里描述的方法将为评估iCM的功能成熟度提供一个有价值的平台，用于心脏重编程研究。

生成Myh6-Cre / Rosa26A-Flox-Stop-Flox-GCaMP3小鼠品系的实验工作流程和转基因小鼠的基因结构如图1所示。在建立小鼠品系的同时，分离幼崽的心脏并在反向荧光显微镜下观察以确认基因型。具有正确基因型的心脏显示Ca2 +通量与跳动同步，可视化为GCaMP3荧光，而在对照心脏中未观察到荧光（图2，视频1和视频2）。分离的NCF将在...

Watch this Scientific Journal Video about In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter at JoVE.com

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

我们在这里描述了Tg（Myh6-cre）1Jmk/J /Gt（ROSA）26Sortm38（CAG-GCaMP3）Hze/J（下面称为αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3）小鼠报告线用于心脏重编程评估的建立和应用。从小鼠菌株中分离出的新生儿心脏成纤维细胞（NCF）转化为诱导心肌细胞（iCM），从而可以方便有效地评估通过钙（Ca2 +）通量对iCM的重编程效率和功能成熟。

使用GCaMP3报告器测量心脏特异性钙通量，对心脏重编程进行体外评估

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

该协议中描述的方法将为评估用于心脏重编程研究的ICM的功能成熟提供有价值的平台。钙通量仅在ICM中观察到，并提供了一种评估ICM功能成熟的方法。这种小鼠品系简化了评估程序，提高了结果的可重复性。该协议是评估ICM在心脏重编程领域的功能成熟的新方法。也可用于评价心肌细胞分化和心功能。首先获得8至10只幼崽，P0至P2幼崽以分离1000万只新生儿心脏成纤维细胞或NCF。通过体温过低深度麻醉幼崽。在荧光显微镜下观察心脏，以确保具有所需基因型的心脏显示钙通量，由GCaMP3指示，心脏跳动。其他基因型不会显示荧光。在隔离心脏后，将它们切成四块，使它们松散地连接在一起。在冰冷的Dulbecco的磷酸盐缓冲盐水或DPBS中，在六厘米的板中彻底洗涤它们几次，以限制分离细胞中的血细胞污染。用8毫升温热的0.25%胰蛋白酶-EDTA在37摄氏度下消化心脏10分钟。丢弃胰蛋白酶上清液，在Hank的平衡盐溶液中加入每毫升0.5毫克的温热型二型胶原酶。彻底涡旋混合物，并在37摄氏度下孵育五分钟。孵育后，再次彻底涡旋混合物，让未消化的组织通过重力沉降下来。将上清液收集在带有5毫升冷成纤维细胞培养基的15毫升锥形管中。用成纤维细胞培养基稀释细胞，使得接种密度约为每立方厘米10至第四个细胞的2至2.5倍。然后将细胞接种在培养皿或盘子中。附着的成纤维细胞在播种后的第二天应具有椭圆形至圆形。在第0天，将NCF接种到大约1到2倍的密度，每立方厘米10到第四个细胞在成纤维细胞培养基中。在第一天，根据需要将培养基替换为每个孔中含有病毒的培养基。在病毒感染后的第2天，24小时，用常规ICM培养基替换含有病毒的培养基。在室温下用倒置荧光显微镜评估钙离子通量。在10X物镜下观察GCaMP3阳性细胞，确保它显示自发细胞在明场通道中跳动。具有正确基因型的心脏显示钙离子通量与跳动同步，可视化为GCaMP3荧光。虽然在对照心脏中没有观察到荧光。分离的NCF在两小时内附着在井上，并在播种后一天显示出椭圆形至圆形。钙离子振荡表明GCaMP3荧光在最大和最小值之间变化，并且这些细胞的钙离子振荡周期性变化。引入IMAP后，由于IMAP培养基处理组具有高功率场钙离子通量的GCaMP3阳性细胞数量增加，跳动簇数显著高于对照组。NCF在隔离后应新鲜准备和健康。快速的心脏隔离，切割和适当的消化时间可以帮助避免细胞活力和状况的降低。该方法为心脏重编程研究人员提供了一种识别功能性饱和ICM的方法。通过多种进一步的评估方法，人们可以更简洁地了解功能性饱和ICM。

Research

JoVE Journal

Medicine

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

多巴酚丁胺诱导的心脏压力测试和监控通过在小鼠心脏导管插入术

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

科研

医学

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

测量心脏自主神经系统（ANS）儿童活动

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

成人大鼠心脏解剖与功能的超声心动图评估

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

小鼠闭胸导管插入术对心功能和压力-容积环的双心室评估

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...